This is a renewal R01 application to exploit previously generated mouse models of germline-reverted HIV Env broadly neutralizing antibodies (gl-bNAbs) and to generate new ones. Developing an effective HIV vaccine is an important goal, as it would facilitate the major public health objective of HIV prevention. The recent identification f bNAbs to HIV Env has shown that such responses are possible. Moreover, animal challenge studies after passive immunization have shown that these bNAbs can be protective. Structural studies have identified the key epitopes at the molecular level, aiding vaccine design. Small animal models potentially can aid HIV vaccine development by allowing one to rapidly assess vaccine candidates and to determine the roadblocks to appropriate gl-bNAb B cell activation and maturation. The hypothesis of this proposal is that mice and B cell lines expressing gl-bNAbs can be useful tools in defining how best to initially stimulate, and then later to train, B cells to generate the desired bNAb responses. Because the problem of poor precursor affinity to antigen is a limitation to all immune responses, knowledge obtained here should be applicable to a variety of vaccine targets. We address these goals through the following Specific Aims: 1) To elicit bNAbs from exisiting gl-bNAb targeted (knock-in) B cells using designer immunogens. Antigens that target naive gl-b12 and gl-4E10 B cells will be used in immunization studies to characterize how best to generate high affinity bNAbs. These studies will be facilitated by the availability of our recently generated germline b12 and 4E10 knock-in mice and specially designed ligands directed to these gl-bNAbs. 2) To generate new gl-bNAb knock-ins for VRC01, 10E8, and either PGT121 or PG9. bNAbs VRC01, 10E8, PG9 and PGT121 see independent, conserved Env epitopes. These potent bNAbs currently appear to be better models than b12 and 4E10 for the development of a good HIV vaccine. Genes for VRC01, 10E8, and either PGT121 or PG9 will be used in the generation of gl-bNAb knock-in mice and in the testing of epitope-scaffold immunogens. These knock-in gl-bNAb mice will be ideal for working out the best ligands and methods for testing in human immunization.